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Estrogen receptors (ERs) play vital roles in the function and remodeling of bone. Their cellular mechanisms can broadly be categorized into those involving direct DNA binding (classical) or indirect DNA binding (non-classical). The generation of non-classical ER knock-in (ERα(-/NERKI) ) mice provides a unique opportunity to define these pathways in bone. We previously demonstrated that ERα(-/NERKI) mice exhibit an osteoporotic phenotype; however, the mechanism(s) for this remain unresolved. Gene expression analyses of cortical bone from ERα(-/NERKI) mice revealed suppression of lymphoid enhancer factor-1 (Lef1), a classic Wnt-responsive transcription factor that associates with β-catenin. Since Wnt signaling is generally considered bone anabolic, this observation leads to the hypothesis that NERKI-induced suppression of Wnt signaling may contribute to the low bone mass phenotype. We generated ERα(-/NERKI) mice crossed with the Wnt-responsive TOPGAL transgenic mouse model and observed significantly less β-galactosidase activity in ERα(-/NERKI) mice, confirming suppression of Wnt activity in vivo. Adenoviral expression of the NERKI receptor using an in vitro cell system resulted in the induction of several secreted antagonists of Wnt signaling. Furthermore, expression of NERKI abrogated Wnt10b-dependent Wnt activation using a lentiviral-mediated reporter assay. Finally, expression of NERKI destabilized β-catenin cellular protein levels and disrupted ER/β-catenin interactions. Collectively, these data suggest the osteoporotic phenotype of ERα(-/NERKI) mice may involve the suppression of Lef1-mediated Wnt signaling through both the stimulation of secreted Wnt inhibitors and/or disruption of normal β-catenin function.

Studies on the pathogenesis of osteoporosis and other metabolic bone diseases would be greatly facilitated by the development of approaches to assess changes in gene expression in osteoblast/osteoprogenitor populations in vivo without the potentially confounding effects of in vitro culture and expansion of the cells. While positive selection to identify a progenitor population in human marrow can be used to select for cells capable of osteoblast differentiation, each of the markers that have been used to identify marrow mesenchymal populations (alkaline phosphatase [AP], Stro-1, CD29, CD49a, CD73, CD90, CD105, CD166, CD44, CD146 and CD271) may be expressed on distinct subsets of marrow mesenchymal cells. Thus, positive selection with one or more of these markers could exclude a possibly relevant cell population that may undergo important changes in various clinical conditions. In the present report, we describe the isolation and characterization of human osteoprogenitor cells obtained by depletion of bone marrow cells of all hematopoietic lineage/hematopoietic stem cells and endothelial/endothelial precursor cells (lin−/CD34/CD31−). The yield of lin−/CD34/CD31− cells from ~10 mL of bone marrow (~ 80 million mononuclear cells) was ~80,000 cells (0.1% of mononuclear cells). While not selected on the basis of expression for the mesenchymal marker, Stro-1, 68% of these cells were Stro-1+. Using linear whole transcriptome amplification followed by quantitative polymerase chain reaction (QPCR) analysis, we also demonstrated that, compared to lin− cells (which are already depleted of hematopoietic cells), lin−/CD34/31− cells expressed markedly lower mRNA levels for the endothelial/hematopoietic markers, CD34, CD31, CD45, and CD133. Lin−/CD34/31− cells were also enriched for the expression of mesenchymal/osteoblastic markers, with a further increase in runx2, osterix, and AP mRNA expression following in vitro culture under osteogenic conditions. Importantly, lin−/CD34/31− cells contained virtually all of the mineralizing cells in human marrow: while these cells displayed robust calcium deposition in vitro, lin−/CD34/31+ cells demonstrated little or no mineralization when cultured under identical osteogenic conditions. Lin−/CD34/31− cells thus represent a human bone marrow population highly enriched for mesenchymal/osteoblast progenitor cells that can be analyzed without in vitro culture in various metabolic bone disorders, including osteoporosis and aging.

Although osteosarcoma represents the most common bone malignancy, the molecular and cellular mechanisms influencing its pathogenesis have remained elusive. Recent evidence has suggested that the Wnt signaling pathway may play a crucial role in osteosarcoma. This study employed a microarray approach to discover novel genes and pathways involved in Wnt signaling in osteosarcoma. We developed a Wnt10b-expressing cell line using the human U2OS osteosarcoma model (U2OS-Wnt10b) and performed microarray and pathway analyses using parental U2OS cells as control. Differential expression of 1,003 genes encompassing 28 pathways was noted. The Wnt, NFκB, and Notch pathways were chosen for further study based on their known importance in bone biology. Known Wnt-responsive genes Axin-2 (4.9-fold), CD44 (2.1-fold), endothelin-1 (4.2-fold) and sclerostin domain containing-1 (43-fold) were regulated by Wnt10b. The proinflammatory cytokines interleukin-1α and tumor necrosis factor-α, known inducers of NFκB, were upregulated both at the transcript and protein level, and NFκB reporter activity was stimulated 3.8-fold, confirming NFκB activation. Interestingly, genes involved in Notch signaling [Notch-1 (2.4-fold) and Jagged-1 (3.1-fold)] were upregulated, whereas the Notch inhibitor, lunatic fringe, was downregulated (8.2-fold). This resulted in the activation of the classic Notch-responsive genes, hairy and enhancer of split-1 (Hes-1; 2.2-fold) and hairy/enhancer-of-split related with YRPW motif-1 (Hey-1; 2.5-fold). A Hey-1 reporter construct was regulated 9.1-fold in U2OS-Wnt10b cells, confirming Notch activation. Interestingly, Wnt3a failed to induce the Notch and NFκB pathways, demonstrating Wnt-specificity. In conclusion, our data demonstrate that Wnt10b, but not Wnt3a, stimulates the NFκB and Notch pathways in U2OS osteosarcoma cells.

Sclerostin is a potent inhibitor of Wnt signaling and bone formation. However, there is currently no information on the relation of circulating sclerostin levels to age, gender, or bone mass in humans. Thus we measured serum sclerostin levels in a population-based sample of 362 women [123 premenopausal, 152 postmenopausal not on estrogen treatment (ET), and 87 postmenopausal on ET] and 318 men, aged 21 to 97 years. Sclerostin levels (mean ± SEM) were significantly higher in men than women (33.3 ± 1.0 pmol/L versus 23.7 ± 0.6 pmol/L, p < .001). In pre- and postmenopausal women not on ET combined (n = 275) as well as in men, sclerostin levels were positively associated with age (r = 0.52 and r = 0.64, respectively, p < .001 for both). Over life, serum sclerostin levels increased by 2.4- and 4.6-fold in the women and men, respectively. Moreover, for a given total-body bone mineral content, elderly subjects (age ≥ 60 years) had higher serum sclerostin levels than younger subjects (ages 20 to 39 years). Our data thus demonstrate that (1) men have higher serum sclerostin levels than women, (2) serum sclerostin levels increase markedly with age, and (3) compared with younger subjects, elderly individuals have higher serum sclerostin levels for a given amount of bone mass. Further studies are needed to define the cause of the age-related increase in serum sclerostin levels in humans as well as the potential role of this increase in mediating the known age-related impairment in bone formation. © 2011 American Society for Bone and Mineral Research.

There is growing interest in the use of mesenchymal stem cells for bone repair. As a major reason for normal bone remodeling is the removal of fatigue microcracks, advances in our understanding of this process may inform approaches to enhance fracture healing. Increasing evidence now indicates that physiological bone remodeling occurs in close proximity to blood vessels and that these vessels carry perivascular stem cells that differentiate into osteoblasts. Similarly, fracture healing is critically dependent on the ingrowth of blood vessels not only for a nutrient supply but also for the influx of osteoblasts. A number of animal and human studies have now shown the potential benefit of bone marrow-derived mesenchymal stem cells in enhancing bone repair. However, as in other tissues, the question of whether these cells improve fracture healing directly by differentiating into osteoblasts or indirectly by secreting paracrine factors that recruit blood vessels and the accompanying perivascular stem cells remains a major unresolved issue. Moreover, CD34+ cells, which are enriched for endothelial/hematopoietic cells, have also shown efficacy in various bone repair models, at least in part due to the induction of angiogenesis and recruitment of host progenitor cells. Thus, mesenchymal and nonmesenchymal stem/progenitor cells are attractive options for bone repair. It is possible that they contribute directly to bone repair, but it is also likely that they express paracrine factors in the appropriate amounts and combinations that promote and sustain the healing process.

Aims Endothelial progenitor cells (EPC) may participate in the repair of injured coronary endothelium. We have recently identified EPC co-expressing the osteoblastic marker osteocalcin [OCN (+) EPC] and found that their numbers are increased in patients with early and late coronary atherosclerosis. The current study was designed to test the hypothesis that early coronary atherosclerosis is associated with the retention of osteogenic EPC within the coronary circulation. Methods and results Blood samples were taken simultaneously from the proximal aorta and the coronary sinus from 31 patients undergoing invasive coronary endothelial function testing. Using flow cytometry, peripheral blood mononuclear cells were analysed for EPC markers (CD133, CD34, KDR) and OCN. The net gradient of EPC was calculated by multiplying the coronary blood flow by the arteriovenous EPC gradient (a negative net gradient indicating retention of EPC). Similarly, serum samples were analysed for stromal cell-derived factor-1 alpha (SDF-1 alpha) and interleukin-8 (IL-8) and their net production calculated. Compared with controls ( n = 17) patients with endothelial dysfunction (ED, n = 14) had a significant net retention of CD34+/CD133−/KDR+/OCN+ EPC [118.38 (0.00, 267.04) vs. −112.03 (838.36, 0.00), P = 0.004]. The retention of OCN (+) EPC correlated with the degree of ED. Patients with ED also showed a net retention of CD34+/CD133−/KDR+ EPC ( P = 0.010). Net production of IL-8 was positive in ED [1540.80 (−300.40, 21744.10)pg/mL] but negative in controls [−3428.50 (−11225.00, 647.48), P = 0.025]. Conclusion Our study demonstrates that patients with early coronary atherosclerosis are characterized by retention of OCN (+) EPC within the coronary circulation, potentially leading to progressive coronary calcification rather than normal repair.

Sex steroids are important regulators of bone turnover, but the mechanisms of their effects on bone remain unclear. Sclerostin is an inhibitor of Wnt signaling, and circulating estrogen (E) levels are inversely associated with sclerostin levels in postmenopausal women. To directly test for sex steroid regulation of sclerostin levels, we examined effects of E treatment of postmenopausal women or selective withdrawal of E versus testosterone (T) in elderly men on circulating sclerostin levels. E treatment of postmenopausal women (n = 17) for 4 weeks led to a 27% decrease in serum sclerostin levels [versus +1% in controls (n = 18), p < .001]. Similarly, in 59 elderly men, we eliminated endogenous E and T production and studied them under conditions of physiologic T and E replacement, and then following withdrawal of T or E, we found that E, but not T, prevented increases in sclerostin levels following induction of sex steroid deficiency. In both sexes, changes in sclerostin levels correlated with changes in bone-resorption, but not bone-formation, markers (r = 0.62, p < .001, and r = 0.33, p = .009, for correlations with changes in serum C-terminal telopeptide of type 1 collagen in the women and men, respectively). Our studies thus establish that in humans, circulating sclerostin levels are reduced by E but not by T. Moreover, consistent with recent data indicating important effects of Wnts on osteoclastic cells, our findings suggest that in humans, changes in sclerostin production may contribute to effects of E on bone resorption. © 2011 American Society for Bone and Mineral Research.

While female mice do not have the equivalent of a menopause, they do undergo reproductive senescence. Thus, to dissociate the effects of aging versus estrogen deficiency on age-related bone loss, we sham-operated, ovariectomized, or ovariectomized and estrogen-replaced female C57/BL6 mice at 6 months of age and followed them to age 18 to 22 months. Lumbar spines and femurs were excised for analysis, and bone marrow hematopoietic lineage negative (lin-) cells (enriched for osteoprogenitor cells) were isolated for gene expression studies. Six-month-old intact control mice were euthanized to define baseline parameters. Compared with young mice, aged/sham-operated mice had a 42% reduction in lumbar spine bone volume/total volume (BV/TV), and maintaining constant estrogen levels over life in ovariectomized/estrogen-treated mice did not prevent age-related trabecular bone loss at this site. By contrast, lifelong estrogen treatment of ovariectomized mice completely prevented the age-related reduction in cortical volumetric bone mineral density (vBMD) and thickness at the tibial diaphysis present in the aged/sham-operated mice. As compared with cells from young mice, lin- cells from aged/sham-operated mice expressed significantly higher mRNA levels for osteoblast differentiation and proliferation marker genes. These data thus demonstrate that, in mice, age-related loss of cortical bone in the appendicular skeleton, but not loss of trabecular bone in the spine, can be prevented by maintaining constant estrogen levels over life. The observed increase in osteoblastic differentiation and proliferation marker gene expression in progenitor bone marrow cells from aged versus young mice may represent a compensatory mechanism in response to ongoing bone loss.

Recent studies have demonstrated an important role for circulating serotonin in regulating bone mass in rodents. In addition, patients treated with selective serotonin reuptake inhibitors (SSRIs) have reduced areal bone mineral density (aBMD). However, the potential physiologic role of serotonin in regulating bone mass in humans remains unclear. Thus we measured serum serotonin levels in a population-based sample of 275 women and related these to total-body and spine aBMD assessed by dual-energy X-ray absorptiometry, femur neck total and trabecular volumetric BMD (vBMD) and vertebral trabecular vBMD assessed by quantitative computed tomography (QCT), and bone microstructural parameters at the distal radius assessed by high-resolution peripheral QCT (HRpQCT). Serotonin levels were inversely associated with body and spine aBMD (age-adjusted R = −0.17 and −0.16, P < .01, respectively) and with femur neck total and trabecular vBMD (age-adjusted R = −0.17 and −0.25, P < .01 and < .001, respectively) but not lumbar spine vBMD. Bone volume/tissue volume, trabecular number, and trabecular thickness at the radius were inversely associated with serotonin levels (age-adjusted R = −0.16, −0.16, and −0.14, P < .05, respectively). Serotonin levels also were inversely associated with body mass index (BMI; age-adjusted R = −0.23, P < .001). Multivariable models showed that serotonin levels remained significant negative predictors of femur neck total and trabecular vBMD, as well as trabecular thickness at the radius, after adjusting for age and BMI. Collectively, our data provide support for a physiologic role for circulating serotonin in regulating bone mass in humans. © 2010 American Society for Bone and Mineral Research

ObjectivesThis study was designed to test whether patients with coronary atherosclerosis have increases in circulating endothelial progenitor cells (EPCs) expressing an osteogenic phenotype.BackgroundIncreasing evidence indicates a link between bone and the vasculature, and bone marrow and circulating osteogenic cells have been identified by staining for the osteoblastic marker, osteocalcin (OCN). Endothelial progenitor cells contribute to vascular repair, but repair of vascular injury may result in calcification. Using cell surface markers (CD34, CD133, kinase insert domain receptor [KDR]) to identify EPCs, we examined whether patients with coronary atherosclerosis had increases in the percentage of EPCs expressing OCN.MethodsWe studied 72 patients undergoing invasive coronary assessment: control patients (normal coronary arteries and no endothelial dysfunction, n = 21) versus 2 groups with coronary atherosclerosis—early coronary atherosclerosis (normal coronary arteries but with endothelial dysfunction, n = 22) and late coronary atherosclerosis (severe, multivessel coronary artery disease, n = 29). Peripheral blood mononuclear cells were analyzed using flow cytometry.ResultsCompared with control patients, patients with early or late coronary atherosclerosis had significant increases (∼2-fold) in the percentage of CD34+/KDR+ and CD34+/CD133+/KDR+ cells costaining for OCN. Even larger increases were noted in the early and late coronary atherosclerosis patients in the percentage of CD34+/CD133−/KDR+ cells costaining for OCN (5- and 2-fold, p < 0.001 and 0.05, respectively).ConclusionsA higher percentage of EPCs express OCN in patients with coronary atherosclerosis compared with subjects with normal endothelial function and no structural coronary artery disease. These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary atherosclerosis.

Introduction: The mechanism(s) by which sex steroids regulate bone turnover in humans are unclear, and recent studies have suggested that follicle-stimulating hormone (FSH) may play an important role in regulating bone resorption. Materials and Methods: Fifty-nine men (median age, 69 yr) underwent suppression of sex steroids using a gonadotropin-releasing hormone (GnRH) agonist and aromatase blocker and were replaced with testosterone (T; 5 mg/d) and estradiol (E; 37.5 μg/d). After assessment of bone resorption markers (serum C-terminal telopeptide of type I collagen [CTX] and TRACP5b), they were randomized to sex steroid deficiency (−T, −E), E alone (−T, +E), T alone (+T, −E), or both (+T, +E) and restudied 3 wk later. Bone marrow aspirates were obtained to isolate osteoblastic, T, and monocytic cells using magnetic-activated cell sorting. Results: Serum CTX and TRACP5b increased significantly (by 71% and 15%, p < 0.01 and < 0.001, respectively) in the −T, −E group, and these increases occurred despite a 60% suppression of serum FSH levels (p < 0.001) caused by the GnRH agonist. There were significant E (but not T) effects on preventing increases in serum CTx and TRACP levels. There was a nonsignificant trend (p = 0.122) for E to suppress RANKL mRNA levels in bone marrow osteoblastic cells. Changes in mRNA levels for other cytokines (TNF-α, interleukin (IL)-1α, IL-1β, IL-1ra, IFN-γ) in bone marrow cells were not significant. Conclusions: E has greater suppressive effects on bone resorption than T, and increased bone resorption after sex steroid deficiency can occur independently of changes in FSH secretion. E effects on bone resorption may be mediated by regulation of RANKL production by osteoblastic cells, although further studies using more highly purified cells may reduce the variability of the mRNA measurements and allow for clearer definition of the mediators of sex steroid action in vivo.

Osteoporosis, a syndrome characterized by thin bones and fractures, has become more prevalent in both women and men. Established therapies for treating this disorder consist primarily of drugs that prevent bone loss, such as the bisphosphonates and selective oestrogen receptor modulators. Although these drugs have been shown to reduce fractures in randomized trials, there is an urgent need for treatments that could lower fracture risk further without additional adverse effects. The introduction of parathyroid hormone (teriparatide), which significantly increases bone mineral density, albeit for a relatively short duration, raised expectations that drugs that stimulate bone formation might cure osteoporosis. After outlining current approaches for treating osteoporosis, this Review focuses on emerging therapeutic opportunities for osteoporosis that are based on recent insights into skeletal physiology. Such novel strategies offer promise not only for reducing age-related bone loss and the associated risk of fractures but also for restoring bone mineral density to healthy levels.

Publisher Summary This chapter reviews the current data on patterns of bone loss with aging and gender that lead to osteoporosis in humans and the mechanisms by which these changes lead to osteoporosis, with particular emphasis on the potential role of immune cytokines in mediating these changes. It also focuses on the key role of estrogen deficiency in bone loss in both women and men, acting both by altering the regulatory cytokines and by other mechanisms. These effects are superimposed on age effects on bone cell function that are independent of both estrogen deficiency and alterations in the production and function of immune cytokines. The past decade has witnessed tremendous advances in our understanding of the pathogenesis of osteoporosis. Newer imaging approaches, including central and peripheral quantitative computed tomography (QCT) and high-resolution peripheral QCT (HRpQCT), have helped to more clearly define the overall pattern of bone mass acquisition and bone loss in both sexes. In addition, fundamental advances in bone biology, such as the discovery and characterization of the receptor-activator of nuclear factor kappa B ligand (RANKL)/RANK/osteoprotegerin (OPG) system, have provided new insights into the mediators of excessive bone resorption following sex steroid deficiency. Concurrent with this greater understanding of bone loss at the clinical and basic levels has been the increasing recognition of the close interactions between bone cells and immune cells, mediated by cytokines produced by both types of cells.

Intermittent parathyroid hormone (PTH) 1–34 treatment stimulates bone formation, but the molecular mechanisms mediating this effect have not been previously studied in humans. Thus, we used magnetic activated cell sorting to isolate hematopoietic lineage negative (lin−)/alkaline phosphatase positive (AP+) osteoprogenitor cells from bone marrow of 20 postmenopausal women treated with PTH (1–34) for 14 days and 19 control subjects. Serum PINP and CTX increased in PTH-treated subjects (by 97% and 30%, respectively, P < 0.001). Bone marrow lin−/AP+ cells from PTH-treated subjects showed an increase in the RANKL/OPG mRNA ratio (by 7.5-fold, P = 0.011) and in the mRNAs for c-fos (a known PTH-responsive gene, by 42%, P = 0.035) and VEGF-C (by 57%, P = 0.046). Gene Set Enrichment Analysis (GSEA, testing for changes in pre-specified pathways) demonstrated that PTH had no effect on osteoblast proliferation, apoptosis, or differentiation markers. However, PTH treatment resulted in a significant decrease (GSEA P-value, 0.005) in a panel of BMP target genes in the lin−/AP+ cells. Our findings thus identify several future directions for studying mechanisms of PTH action in humans. First, given the increasing evidence that PTH induces angiogenesis, the role of increased VEGF-C production by bone marrow osteoprogenitor cells in mediating this effect and the anabolic response to PTH warrants further study. Second, while the observed inhibition of BMP target gene expression by PTH is not consistent with the anabolic effects of PTH on bone and requires further validation, these data do generate the hypothesis that an inhibition of BMP signaling by PTH may, over time, limit the availability of mature osteoblasts on bone surfaces and thereby contribute to the observed waning of the anabolic response to PTH.

Context: Intermittent PTH treatment stimulates bone formation, but the mechanism(s) of this effect remain unclear. Sclerostin is an inhibitor of Wnt signaling, and animal studies have demonstrated that PTH suppresses sclerostin production. Objective: The objective of the study was to test whether intermittent PTH treatment of postmenopausal women alters circulating sclerostin levels. Design: Prospective study. Setting: The study was conducted at a clinical research unit. Participants and Interventions: Participants included 27 postmenopausal women treated with PTH (1-34) for 14 d and 28 control women. Main Outcome Measures: Serum sclerostin levels were measured. Results: Circulating sclerostin levels decreased significantly in the PTH-treated subjects, from (mean ± SEM) 551 ± 32 to 482 ± 31 pg/ml (−12.7%, P < 0.0001) but did not change in the control women (baseline, 559 ± 34 pg/ml; end point, 537 ± 40 pg/ml, P = 0.207; P = 0.017 for difference in changes between groups). Bone marrow plasma was obtained in a subset of the control and PTH-treated subjects (n = 19 each) at the end of the treatment period, and marrow plasma and peripheral serum sclerostin levels were significantly correlated (R = 0.64, P < 0.0001). Marrow plasma sclerostin levels were 24% lower in PTH-treated compared with control women, but perhaps due to the smaller sample size, this difference was not statistically significant (P = 0.173). Conclusions: Circulating sclerostin levels correlate with bone marrow plasma levels and are reduced by intermittent PTH therapy in postmenopausal women. Further studies are needed to assess the extent to which decreases in sclerostin production contribute to the anabolic skeletal response to PTH.

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

International audience; Both estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARgamma) regulate bone metabolism, and because steroid receptor coactivator (SRC)-2 (TIF-2) enhances ER and PPARgamma activity, we examined the consequences of deletion of SRC-2 on bone using SRC-2 knock out (KO) mice. Loss of SRC-2 resulted in increased bone mass, with SRC-2 KO mice having 80% higher trabecular bone volume as compared with wild type mice. SRC-2 KO mice also had a marked decrease (by 50%) in bone marrow adipocytes. These data suggested that marrow precursor cells in the SRC-2 KO mice may be resistant to the inhibitory effects of endogenous PPARgamma ligands on bone formation. Consistent with this, compared with cultures from wild type mice, marrow stromal cultures from SRC-2 KO mice formed significantly more mineralized nodules (by 3-fold) in the presence of the PPARgamma agonist, rosiglitazone. Using chromatin immunoprecipitation analysis, we demonstrated that in bone marrow stromal cells, loss of SRC-2 leads to destabilization of the transcription complex at the peroxisome proliferator response elements of a number of PPARgamma target genes, resulting in an overall decrease in the expression of adipocyte-related genes and a marked decrease in adipocyte development. Using ovariectomy with or without estrogen replacement, we also demonstrated that SRC-2 KO mice were partially resistant to the skeletal actions of estrogen. Collectively, these findings indicate that loss of SRC-2 leads to partial skeletal resistance to the ER and PPARgamma, but resistance to PPARgamma is dominant, leading to increased bone mass. Modulating SRC-2 action may, thus, represent a novel therapeutic target for osteoporosis.

Plastic adherent bone marrow stromal cells have become synonymous with skeletal stem cells, and perhaps rightfully so, as these cells have been extremely well characterized over the past four decades, since their original description by Friedenstein. However, although this cell population is useful as an experimental model of precursors for osteoblasts and other mesenchymal lineages, the precise role of bone marrow stromal cells in bone remodeling, fracture repair, or repair of non-skeletal tissues remains unclear. Moreover, there is a conceptual problem in terms of postulating that these cells are osteoblast precursors at sites of bone remodeling on trabecular surfaces adjacent to red marrow and yet having to posit potentially entirely different mechanisms for the origins of osteoblasts at sites of cortical bone remodeling distant from red marrow. Thus, the identification and characterization in recent years of non-adherent stem and osteoprogenitor cells in the bone marrow, of similar cells in the peripheral circulation, and of stem/osteoprogenitor cells arising either from the perivascular compartment (pericytes) or within the developing vascular wall itself, has suggested alternative candidate cell populations that may help to resolve the problem of postulating different mechanisms of remodeling in trabecular versus cortical bone. When coupled with our evolving understanding of the bone remodeling compartment (BRC), a closed cavity penetrated by capillaries which appears to be the site of remodeling in both trabecular and cortical bone, it is likely that our conceptual understanding of the fundamental mechanisms of bone remodeling will need to be modified. J. Cell. Biochem. 103: 393–400, 2008. © 2007 Wiley-Liss, Inc.

We recently identified circulating osteoblastic cells using antibodies to osteocalcin (OCN) or alkaline phosphatase (AP). We now provide a more detailed characterization of these cells. Specifically, we demonstrate that 46% of OCN positive (OCNpos) cells express AP, and 37% also express the hematopoietic/endothelial marker, CD34. Using two different anti-OCN antibodies and forward/side light scatter characteristics by flow cytometry, we find that OCNpos cells consist of two distinct populations: one population exhibits low forward/side scatter, consistent with a small cell phenotype with low granularity, and a second population has higher forward/side scatter (larger and more granular cell). The smaller, low granularity population also co-expresses CD34, whereas the larger, more granular cells are CD34 negative. Using samples from 26 male subjects aged 28 to 68 years, we demonstrate that the concentration of circulating OCNpos cells increases as a function of age (R = 0.59, P = 0.002). By contrast, CD34pos cells tend to decrease with age (R = −0.31, P = 0.18); as a consequence, the ratio of OCNpos:CD34pos cells also increases significantly with age (R = 0.54, P = 0.022). These findings suggest significant overlap between circulating cells expressing OCN and those expressing the hematopoietic/endothelial marker, CD34. Further studies are needed to define the precise role of circulating OCNpos cells not only in bone remodeling but rather also potentially in the response to vascular injury.

ER alpha acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ER alpha signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. INTRODUCTION: Estrogen receptor alpha (ER alpha) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ER alpha knock-in [NERKI]) with heterozygote ER alpha knockout mice and studied the resulting female ER alpha(+/+), ER alpha(+/NERKI), and ER alpha(-/NERKI) mice. The only ER alpha present in ER alpha(-/NERKI) mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ER alpha(+/NERKI) mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. MATERIALS AND METHODS: BMD was measured using DXA and pQCT at 3 months of age (n = 46-48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 microg/day), or ovariectomy + estradiol (1.0 microg/day; n = 10-12/group) and restudied 60 days later. RESULTS AND CONCLUSIONS: At 3 months of age, both the ER alpha(+/NERKI) and ER alpha(-/NERKI) mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ER alpha(-/NERKI) mice were the opposite of those in ER alpha(+/+) mice. Relative to sham mice, ovariectomized ER alpha(-/NERKI) mice gained more bone (not less, as in ER alpha(+/+) mice), and estrogen suppressed this increase (whereas augmenting it in ER alpha(+/+) mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ER alpha(-/NERKI) versus ER alpha(+/+) mice. Collectively, these data show that alteration of the balance between classical and nonclassical ER alpha signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ER alpha signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.

BACKGROUND: Estrogen is known to have important effects on both reproductive and non-reproductive tissues. Moreover, there is increasing interest in developing compounds that may have selective effects on bone versus reproductive tissues. METHODS: Since mouse models are often used in these studies, we administrated increasing doses of estradiol (E2) (0 to 500 microg/kg/day) by slow release pellets to ovariectomized 6-month-old C57BL/6 mice and assessed skeletal and uterine responses following 2 months of treatment. RESULTS: The mice lost bone at multiple sites following ovariectomy (OVX); however, while the lowest E2 dose of 5 microg/kg/day completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis), it had no stimulatory effects on the uterus. Higher doses of E2 resulted in further increases in bone mineral density, with eventual stimulation of the uterus at a dose of 40 microg/kg/day. By contrast, when 3-month-old C57BL/6 mice were administered the same doses of E2 and studied after 1 month, the 5 microg/kg/day dose resulted in uterine hypertropy, but was not able to prevent loss of cancellous bone. CONCLUSIONS: Thus these results (i) provide data on the dose-response for the effects of E2 on mouse bone and (ii) indicate that the relative effects of E2 on bone versus the uterus are highly dependent on the particular experimental conditions used. This issue needs to be considered in evaluating agents with potential 'selective' effects on bone versus reproductive tissues.

Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen (E) action in many tissues, but its role in mediating E effects on bone is unknown. Thus, we assessed the skeletal response to ovariectomy (ovx) and E replacement in SRC-1 knockout (KO) mice compared with wild-type (WT) littermates. Bone mineral density was measured by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at baseline and after 2 months of sham surgery, ovx, or ovx plus E replacement. Microcomputed tomography and bone histomorphometry were also performed at the end of the study. Both WT and SRC-1 KO mice lost bone at multiple sites after ovx; however, although an estradiol (E(2)) dose of 10 microg/kg.d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis) in the WT mice, it was entirely ineffective in preventing cancellous bone loss at these sites in the SRC-1 KO mice. This E(2) dose was, however, equally effective on cortical bone in the tibia in the SRC-1 KO and WT mice. Moreover, a 4-fold higher dose of E(2) was able to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice. These findings establish that, in mice, loss of SRC-1 leads to skeletal resistance to E predominantly in cancellous bone.